Method For Separating Cells Using Immunorosettes and Magnetic Particles

ABSTRACT

A concussive injury micronutrient formulation is provided and the formulation comprises vitamin A, vitamin E, natural mixed carotenoids, vitamin C, vitamin D, coenzyme Q10, alpha lipoic acid, N-acetyl cysteine, acetyl L-camitine (fumarate), vitamin B, folic acid, calcium, magnesium, selenium, chromium, biotin, zinc, and omega-3 fatty acids. The formulation is to be taken orally by humans and taken twice a day.

RELATED APPLICATION

This application is related to U.S. Provisional Application Ser. No.61/962,802, entitled “Micronutrient Formulations for Concussive BrainInjuries” which was filed on Nov. 18, 2013.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to micronutrient formulations fortreatment and/or prevention of traumatic brain injuries (TBI) such asconcussions.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides for a micronutrientformulation comprising vitamin A, vitamin E, natural mixed carotenoids,vitamin C, vitamin D, coenzyme Q10, alpha-lipoic acid, N-acetylcysteine, acetyl L-carnitine (fumarate), vitamin B, folic acid, calcium,magnesium, selenium, chromium, biotin, zinc, and omega-3 fatty acidswherein said formulation is designed for treatment and prevention ofconcussions.

In another embodiment, the present invention relates to a micronutrientformulation comprising: vitamin A, vitamin E, vitamin C, vitamin D,coenzyme Q10, N-acetyl cysteine, acetyl L-carnitine, vitamin B, folicacid, calcium, magnesium, selenium, chromium, biotin, zinc, sodium,potassium, carbohydrates wherein said formulation is designed to treatand prevent concussions.

DETAILED DESCRIPTION OF THE INVENTION

As required, detailed embodiments of the present invention are disclosedherein; however, it is to be understood that the disclosed embodimentsare merely exemplary of the invention that may be embodied in variousforms. The figures are not necessarily to scale, some features may beexaggerated to show details of particular components. Therefore,specific structural and functional details disclosed herein are not tobe interpreted as limiting, but merely as a basis for the claims and asa representative basis for teaching one skilled in the art to variouslyemploy the present invention.

The specific example below will enable the invention to be betterunderstood. However, they are given merely by way of guidance and do notimply any limitation.

Traumatic brain injury (TBI) occurs when sudden trauma causes damage tothe brain. TBI can occur with or without penetrating head injury. TBIwithout penetrating head injury is known as a concussive brain injurythat may be expressed as a mild, moderate or severe form. A concussionoccurs when the brain is violently rocked back and forth within theskull following a blow to the head or neck, such as observed invehicular accidents and contact sports like football, or when the victimis in close proximity to a concussive blast pressure wave, such as inindustrial or combat-related circumstances. Because there is nopenetration of the skull, concussive brain injury may be difficult toidentify after the inciting event. Therefore prevention strategies andstandardized treatment guidelines remain elusive. This patent exploitsan understanding of the mechanisms of cellular damage, the biologicalevents that impact long-term neurologic outcomes, and the application ofantioxidant science to develop a rational strategy for reducing theprogression of TBI.

Both experimental and human studies suggest that increased levels ofoxidative stress from excessive production of free radicals (1-5),products of uncontrolled acute and chronic inflammation (6-12) andrelease of a neurotoxic substance, glutamate, in the brain are involvedin the progression of damage following concussive injury, and contributeto the initiation and progression of brain damage. These observationsare supported by the fact that individual antioxidants (known toneutralize free radicals, reduce inflammation, and prevent the releaseand toxicity of glutamate), have reduced damage when administered eitherbefore or soon after concussion (2, 4, 13, 14). The expression ofoncogene proteins, c-myc and c-fos, is elevated in rat brains afterconcussion (15, 16), and cortical expression of nuclear factor kappa B(NF-kappa B) is elevated in human contused brain (17). The fact thatantioxidants and their derivatives reduce expression of c-myc oncogene(18) and activation of NF-kappa B (19), further suggests their value inreducing brain damage following concussive injury. This applicationpresents the scientific rationale and evidence that a specificformulation of multiple micronutrients, including dietary and endogenousantioxidants and glutathione-elevating agents, may prevent and improvestandard therapy for TBI, and defines the composition, dosage, and doseschedule that most effectively impacts the acute- and long-termmanagement of concussive brain injury.

Incidence and Cost of TBI

Annually, about 1.4 million people in the U.S. suffer from TBI, of which1.1 million are treated and released from an emergency department,235,000 are hospitalized, and 50,000 die (20). Among children ages 0 to14 years, concussive brain injuries cause 2685 deaths, 37,000hospitalizations and 435,000 emergency room visits each year. The CDCInjury Center has estimated that about 3.8 million sports- andrecreation-related concussions occur annually in the U.S. In total, atleast 5.3 million Americans who suffered from a TBI have long-term orlife-long need for assistance to perform the routine activities of dailyliving. The cost per year per person with mild TBI is about $32,000;with moderate to severe TBI, it is from about $268,000 to more than$408,000. In 2000, the direct medical costs and indirect costs such aslost productivity because of concussive brain injury were estimated tobe $60 billion in the U.S. (21). In the military, this is becoming aserious issue as about 30% of all soldiers with combat-related injuriesseen at Walter Reed Army Medical Center from 2003 to 2005 sustained aconcussive blast. By 2011, about 320,000 soldiers who served in Iraq andAfghanistan suffered a TBI.

Symptoms and Consequences of Concussive Injury

Symptoms of concussive injury can be mild, moderate or severe, dependingupon the extent of damage to the brain. Individuals with a mild TBI mayremain conscious or may become unconscious for a few second or minutes,and may exhibit headache, confusion, lightheadedness, dizziness, blurredvision, tinnitus (ringing in the ears), fatigue, alterations in sleepingpatterns, mood changes, trouble with memory, concentration, andthinking. Individuals with a moderate or severe TBI can sufferadditional symptoms including a headache that gets worse or remainspersistent, repeated vomiting or nausea, convulsions or seizures, aninability to awaken from sleep, dilation of one or both pupils of theeyes, slurred speech, weakness or numbness in the extremities, loss ofcoordination, and increased confusion, restlessness or agitation. Thisimpact on long-term quality of life may affect individuals with TBI forextended periods of time.

Despite evolutionary changes in protective equipment, concussiveinjuries remain a major health risk for those who engage in potentiallyviolent sports (22, 23) or hazardous occupations, including militarycombat. Cerebral concussion is a type of TBI that is normally producedby acceleration and deceleration of the head. It can also happen duringthe rapid displacement and rotation of the cranium after peak headacceleration and momentum transfer by helmet impacts (24). It ischaracterized by a sudden brief impairment of consciousness, paralysisof reflex activity and loss of memory. Sport-related concussions havebeen classified into simple and complex concussions (recommendation ofthe Second International Conference on Concussion in Sport, 2005).Athletes who are slow to recover (i.e., greater than 10 days) areclassified having complex concussions. Brain deformation may occur afterthe primary head acceleration (24). Damage to the mid brain correlatedwith memory and cognitive problems after concussion. The majordepression commonly observed after concussion contributes to impairmentof memory, processing speed, verbal memory and executive function(25-27). An early onset of dementia may be initiated by repetitivecerebral concussions in professional football players (28, 29). Balancedisorders are also considered one of the major health problemsassociated with the TBI (29, 30). Current efforts on reducing the impactof concussion have focused on the development of physical protection.Indeed, the introduction of newer football helmets appears to lower therisk of concussion by about 10-20% (30).

Extensive evaluation of the relationship between post-traumatic stressdisorder (PTSD) and concussive brain injury has been demonstrated in tworecent epidemiologic studies in U.S. soldiers returning from Iraq andAfghanistan (31, 32). Among 2525 soldiers, 4.9% reported injury withloss of consciousness, 10.3% reported injuries with altered mentalstatus, and 17.2% reported other injuries during deployment. Among thosewho reported loss of consciousness, the incidence of PTSD was about43.9%. Among those reporting altered mental status, it was 27.3%, andamong those reported other injuries, it was 16.2%. In contrast, amongthose soldiers reporting no injury in combat, the incidence of PTSD wasonly 9.1%. This patent describes a novel biological protection strategyto reduce the acute and long-term impact of concussion and would becomplimentary to existing physical protection. The development of thisstrategy is based on the biochemical events that initiate damage andthat contribute to the progression of damage following concussion.

Biochemical Events Contributing to Damage Progression after TBI

Both animal and human studies show that TBI causes a significant loss ofcortical tissue at the site of injury (primary damage). This is followedby secondary damage involving increased oxidative damage, mitochondrialdysfunction, release of predominantly pro-inflammatory cytokines andtoxic levels of glutamate leading to cognitive impairment andneurological dysfunction. Therefore, attenuation of these biochemicalevents may help to reduce the onset and progression of damage.

Increased oxidative stress due to production of excessive amounts offree radicals derived from oxygen and nitrogen occur after concussions(33-36). The extent of oxidative damage appeared to be directlyproportion to the severity of TBI (37). In rats, there appears to be aclose relationship between the degree of oxidative stress and severityof brain damage following concussive brain injury as evidenced by thehigh levels of malondialdehyde (lipid peroxidation marker of oxidativedamage) and low levels of ascorbate (antioxidant vitamin) (38). TBIinduced peroxynitrite-mediated oxidative damage to mitochondrialfunction that precedes neuronal loss in the brain. The oxidation ofnitric oxide (NO) forms peroxynitrite. Animal studies show that TBIincreased NO production that impairs mitochondrial function byinhibiting cytochrome oxidase (39). Cytochrome oxidase is a key enzymeneeded to generate energy. Thus, the energy level in tissue decreasedafter concussive injury and this may interfere with repair processes.TBI also increased inducible nitric oxide synthase (iNOS) activity thatcontributes to neurological deficits by generating excessive amounts ofNO (40). In a rat model of traumatic injury (unilateral moderatecortical contusion), increased oxidative damage occur as early as 3hours following TBI that adversely affects synaptic function andneuronal plasticity, and thereby, enhances cognitive dysfunction (41).In another model of TBI (fluid percussion brain injury in rat), it wasobserved that levels of oxidative stress increased in the cortex of thebrain when measured one and three months following injury.

Human studies also confirm the role of oxidative stress in theprogression of TBI. F2-isoprostane is a marker of lipid peroxidation,whereas neuron-specific enolase (NSE) is considered a marker of neuronaldamage. The levels of F2-isoprostane and NSE increased in the cerebralspinal fluid (CSF) following concussive brain injury in children andinfants (42). The levels of the antioxidants ascorbate and glutathionedecreased in the CSF of children and infants following TBI (43). It hasalso been reported that the levels of beta-amyloid fragment (A beta-42)increased in the CSF of patients after severe concussive brain injury(44). This peptide has been implicated in causing neuronal damage inpatients with Alzheimer's disease (45-48).

Mitochondrial Dysfunction in Concussive Brain Injury

Increased oxidative stress contributes to the mitochondrial dysfunctionthat plays a central role in causing cognitive impairment and eventuallycell death following TBI (49, 50). In a rat model, several mitochondrialproteins involved in bioenergetics were oxidized following injurycausing mitochondrial dysfunction (51). In another rat model, it wasfound that the activity of the mitochondrial enzymes decreased, whileacid-base balance was disrupted and levels of oxidative stress increasedin the blood following concussions. These changes contribute to theseverity of brain injury. Generally, oxidative stress-inducedmitochondrial dysfunction is observed one to three hours after TBI,suggesting the importance of early intervention to reduce the oxidativestress (52). There is a direct link between energy metabolism andN-acetylaspartate. In a human clinical study involving 14 patients (6patients with diffuse brain injury and 8 with focal brain lesions), itwas observed that reduction in the brain levels of n-acetylaspartate inthe absence of ischemic insult reflected mitochondrial dysfunction (53).

Increased Inflammation in TBI

Following TBI, brain cells generate excessive amounts ofpro-inflammatory cytokines, prostaglandins, reactive oxygen species,complement proteins and adhesion molecules that are highly toxic toneurons (54-58). Evidence of inflammation is also found by theinfiltration and accumulation of polymorphonuclear leukocytes.Pro-inflammatory cytokines increased the expression of the enzymeinducible nitric oxide synthase (iNOS) producing excessive amounts of NOthat can become oxidized to form peroxynitrite and contributes to thepathogenesis of concussive brain injury (59-61). An inhibitor of iNOSprovided neuroprotection against damage produced by peroxynitrite (62).The pro-inflammatory cytokine interleukin-6 (IL-6) is elevated inpatients with acute TBI, and a significant relationship exists betweenthe severity of TBI and the IL-6 level (63). In a clinical studyinvolving 75 patients with moderate to severe concussions, the role ofcytokines and lipids relative to 30-day mortality was evaluated. Theresults showed that the levels of cytokines (IL-6 and IL-8) increasedand lipid decreased in all patients, especially non-survivors, comparedto those in the healthy control group (64). In addition, severe TBI ininfants and children (N=36) increased the levels of pro-inflammatorycytokines (IL-1beta, IL-6 and IL-12p70) and anti-inflammatory cytokines(IL-10) and chemokines (IL-8 and MIP-1alpha) compared to controls.

In animal models, the levels of inflammation markers such as iNOS andcyclooxygenase 2 activity, and markers of oxidative stress (loss ofglutathione and oxidized: reduced glutathione ratio, 3-nitrotyrosine,and 4-hydroxynonenal) increased after concussions (65). Motorperformance and spatial memory acquisition were improved in geneticallyprotected mice compared to control mice after subjecting them to a brainimpact model of TBI (66). The role of pro-inflammatory cytokines in theprogression of damage following concussive injury is further supportedby the fact that inhibitors of these cytokines improved neuronal lossand cognitive dysfunction.

Increased Glutamate Release in TBI

The excitatory amino acids (glutamate and aspartate) play a significantrole in the progression of injury following concussions (67). Excessiveamounts of glutamate in the extracellular space may cause swelling,edema and eventually cell death. In a clinical study involving 80patients with severe head injury, it was observed that the levels ofexcitatory amino acids increased and enhanced neuronal damage (68). Inpatients with focal and diffuse brain injury, the levels of glutamatewere elevated in both cerebrospinal fluid and extracellular space (69).In another clinical study, it was found that patients who died of theirhead injury had higher levels of dialysate glutamate and aspartatecompared to those who recovered (67). It was also observed that thelevels of adenosine and glutamate were elevated in the ventricular CSFin 27 children with severe TBI compared to 21 children withoutconcussive brain injury (70). The involvement of glutamate in theprogression of damage following concussion is further suggested by thefact that administration of antagonists to glutamate release improvedmotor function and cognitive dysfunction in animal models of TBI (71,72).

Treatment of Concussive Brain Injury with Antioxidants

Since increased oxidative stress, chronic inflammation and glutamaterelease are involved in the development and progression of neurologicaldeficits, and since antioxidants are known to reduce these factors, anovel biological protection strategy of daily supplementation withappropriate types of multiple micronutrients including dietary andendogenous antioxidants can be expected to reduce the risk ofneurological dysfunction.

Resveratrol, a phenolic antioxidant, administered immediately after TBIreduced oxidative damage and lesion volume in rats (73, 74). Edaravone,an FDA approved drug, reduced oxidative damage by neutralizing freeradicals after concussive injury in humans (75). Superoxide dismutaseimproved TBI-induced mitochondrial dysfunction in mice (76). Treatmentwith alpha-lipoic acid reduced markers of pro-inflammatory cytokines andoxidative stress, and improved histological changes in the brain,preserved blood-brain-barrier permeability and reduced edema followingTBI in animals (77). Administration of N-acetylcysteine providedneuroprotection in animal models following concussion by reducingmarkers of pro-inflammatory cytokines and adhesion molecules (78).Melatonin, a pineal hormone exhibiting antioxidant activity, protectedagainst TBI-induced damage (79). Dietary supplementation with vitamin Eor curcumin protected the brain against damage after concussive injuryby reducing the biochemical changes involved in synaptic plasticity andcognitive function (14, 80, 81). Vitamin E also inhibited the releaseand toxicity of glutamate (82, 83). Dietary supplementation of omega-3fatty acids in an animal model protected against TBI-induced reducedsynaptic plasticity and cognitive impairment (84). In addition to thedata relative to oxidative damage, there is substantial evidence thatdietary and endogenous antioxidants and antioxidants derived from herbsand fruits and vegetables inhibit inflammation (85-97). Increasedpro-inflammatory stimuli and oxidative stress cause brain tissue torelease excessive amounts of glutamate after TBI, which contributes toloss of neurons (98). Release of glutamate was blocked by vitamin E(98). Both vitamin E (83) and coenzyme Q10 (82) also protect againstglutamate-induced neurotoxicity in cell culture models.

Rationale for Employing Multiple Micronutrients in TBI

While laboratory studies in animal models show that supplementation witha single micronutrient such as an antioxidant may protect the brainagainst TBI-induced biochemical and structural damage, similar clinicalstudies have produced inconsistent results. It is well established thatthe internal oxidative environment in these populations is high, andthat an individual antioxidant when oxidized acts as a pro-oxidant.Therefore, administration of a single antioxidant under high riskconditions may produce pro-oxidant rather than antioxidant effects.Because increased levels of oxidative stress and chronic inflammation,as well as enhanced release of glutamate follow TBI, oralsupplementation with appropriate multiple micronutrients includingdietary and endogenous antioxidants as an adjunct to standard therapycan be expected to reduce the risk of late adverse effects on brainfunction. In addition, when administered in a concussion-proneenvironment, but before the event occurs, they may prevent or reduce thelevel of initial damage.

For the purpose of uniqueness in this patent application, specificcomprehensive combinations of antioxidants and glutathione-elevatingagents is mandatory because their mechanisms of action and distributionat cellular and organ levels differ, their cellular and organenvironments (oxygenation, aqueous and lipid components) differ, andtheir affinity for various types of free radicals differs. For example,beta-carotene (BC) is more effective in quenching oxygen radicals thanmost other antioxidants (99). BC can perform certain biologicalfunctions that cannot be produced by its metabolite vitamin A, and viceversa (100, 101). It has been reported that BC treatment enhances theexpression of the connexin gene which codes for a gap junction proteinin mammalian fibroblasts in culture, whereas vitamin A treatment doesnot produce such an effect (101). Vitamin A can induce differentiationin certain normal and cancer cells, whereas BC and other carotenoids donot (102, 103). Thus, BC and vitamin A have, in part, differentbiological functions.

The gradient of oxygen pressure varies within cells. Some antioxidants,such as vitamin E, are more effective as quenchers of free radicals inreduced oxygen pressure, whereas BC and vitamin A are more effective inhigher atmospheric pressures (104). Vitamin C is necessary to protectcellular components in aqueous environments, whereas carotenoids andvitamins A and E protect cellular components in lipid environments.Vitamin C also plays an important role in maintaining cellular levels ofvitamin E by recycling vitamin E radical (oxidized) to the reduced(antioxidant) form (105). Also, oxidative DNA damage produced by highlevels of vitamin C could be protected by vitamin E. Oxidized forms ofvitamin C and vitamin E can also act as radicals; therefore excessiveamounts of any one of these forms, when used as a single agent, could beharmful over a long period of time.

The form of vitamin E used is also important in any clinical trial. Ithas been established that d-alpha-tocopheryl succinate (alpha-TS) is themost effective form of vitamin both in vitro and in vivo (106, 107).This form of vitamin E is more soluble than alpha-tocopherol and enterscells more readily, and, therefore, is expected to cross the blood-brainbarrier more efficiently in TBI.

Glutathione is one of the body's most important antioxidants. However,oral supplementation of this substance failed to significantly increaseplasma levels of glutathione in human subjects (108) suggesting thatthis tripeptide is completely hydrolyzed in the gastrointestinal tract.N-acetylcysteine and alpha-lipoic acid increase the cellular levels ofglutathione by different mechanisms and can be effectively combined in amultiple micronutrient preparation. Selenium is a co-factor ofglutathione peroxidase that also increases the intracellular level ofglutathione. In addition, R-alpha-lipoic acid and acetyl-L-carnitinetogether promoted mitochondrial biogenesis whereas no effect wasobserved when these antioxidants were used individually (109).

Other endogenous antioxidants, such as coenzyme Q₁₀, also have potentialvalue in prevention and adjunctive treatment of TBI. Since mitochondrialdysfunction occurs in patients with concussive injury and since coenzymeQ₁₀ is needed for the generation of ATP by mitochondria, thisantioxidant is essential to improve the mitochondrial function.Ubiquinol (coenzyme Q₁₀) scavenges peroxy radicals faster thanalpha-tocopherol (110) and like vitamin C, can regenerate vitamin E in aredox cycle (111). Coenzyme Q₁₀ administration has also been shown toimprove clinical symptoms in patients with mitochondrialencephalomyopathies (112).

Antioxidant micronutrients can reduce the risk of neurologicaldysfunction in other conditions such as Alzheimer's and Parkinson'sdisease, and this neuroprotective value is directly relevant toconcussive brain injury and the common late adverse effects.Beta-amyloid fragments that are associated with neurodegeneration inAlzheimer's disease mediate their action by free radicals (113). This issupported by the fact that vitamin E protects neuronal cells in cultureagainst beta-amyloid-induced toxicity (114). Vitamin E at a dose of2,000 IU per day can produce beneficial effects in patients withAlzheimer's disease (115). Patients consuming antioxidants also showedreduced risk of vascular dementia and slower decline of cognitivefunction in cases of dementia and Alzheimer's disease (116).Prostaglandin E2, a product of inflammatory reactions, is very toxic tomature neurons, and a mixture of antioxidants reduces this toxicity(117). Furthermore, glutathione deficiency has been consistently foundin autopsied brain samples from patients with neurological conditionssuch as Alzheimer's disease (118) and Parkinson disease (119, 120),again demonstrating the relevance of employing micronutrients thatincrease glutathione for TBI.

Micronutrient Formulations for Concussive Brain Injury

The formulations for concussive brain injury can be placed in thefollowing forms capsules, pills, beverages, powders, chewables (e.g.gummies), dissolvables (e.g oral wafers and disks), solids (e.g. bars),functional foods (e.g. yogurts and creams), sprays, inhaled aerosols,suspensions, liposomes, extended release technologies, and otherabsorbable forms such as skin patches and injectables.

These comprehensive formulations are intended for individuals who havealready suffered initial or repeated TBI from contact sports, accidents,dangerous occupational environments or military combat, or for those whomay likely suffer such an event because of exposure to these high riskcircumstances. The cognitive dysfunction and other neurologicalabnormalities may be noted soon after concussions or may not be evidentuntil several years later.

Examples of Micronutrient Formulations Example 1

A formulation for “youth”, for example ages 9-13 years, is provided in abottled form (two capsules taken twice per day), liquid form (one packetdissolved in liquid twice per day), beverage bottle (8-12 ounces) or barform (one bar consumed twice per day) and is recommended (but notrequired) to be taken year round by young athletes.

One serving of the daily consumption (two servings per day) is comprisedof: vitamin A (retinyl palmitate, fish oil, or natural mixedcarotenoids) 1500 IU, vitamin E (both d-alpha-tocopherol 50 IU andd-alpha tocopheryl succinate 50 IU, vegetable products, naturaltocopheryl, or wheat germ products),

vitamin C (calcium ascorbate, citrus, rose, or berry products) 250 mg,vitamin D (cholecalciferol-D3, or fish oils) 200 IU,coenzyme Q10 (ubiquinone) 45 mg,R-alpha-lipoic acid (R+ or R−) 15 mg,N-acetyl cysteine 25 mg,acetyl L-carnitine (fumarate) 50 mg,vitamin B1-(thiamine mononitrate or yeast sources) 2 mg,vitamin B2-(riboflavin or yeast sources) 2.5 mg,vitamin B3-(niacinamide or yeast sources) 5 mg,vitamin B6-(pyridoxine hydrochloride or yeast sources) 2.5 mg,folic acid (folate, yeast source, or liver sources) 200 mcg,vitamin B12-(cyanocobalamin or yeast source) 5 mcg,biotin (d-biotin or liver sources) 100 mcg,vitamin B-5 (pantothenic acid, D-calcium pantothenate, pantothenate,yeast sources, rice bran, or liver sources) 5 mg,calcium (citrate, ascorbate, plant based sources, or lactate) 125 mg,magnesium (citrate, lactate, or natural forms) 62.5 mg,selenium (seleno-L-methionine or natural forms) 50 mcg,zinc (glycinate or natural forms) 7.5 mg, andchromium (picolinate or natural forms) 25 mcg.

Example 2

A formulation for “adults”, for examples ages 14 years and older, isprovided in a bottled form (three capsules plus one fatty acid gel-captaken twice per day), liquid form (one packet dissolved in liquid twiceper day), beverage bottle (8-32 ounces), or bar form (one bar consumedtwice per day) and is recommended (but not required) to be taken yearround by those engaging in contact sports, working in hazardousoccupations, or those in the active military.

The daily consumption is comprised of:

vitamin A (retinyl palmitate, fish oil, or natural mixed carotenoids)3000 IU,vitamin E (both d-alpha-tocopherol 100 IU and d-alpha tocopherylsuccinate 300 IU, vegetable products, natural tocopheryl, or wheat germproducts),vitamin C (calcium ascorbate, citrus, rose, or berry products) 1000 mg,vitamin D (cholecalciferol-D3 or fish oils) 800 IU,coenzyme Q10 (ubiquinone) 120 mg,alpha-lipoic acid (R+ or R−) 90 mg,N-acetyl cysteine 200 mg,acetyl L-carnitine (fumarate) 150 mg,vitamin B1 (thiamine mononitrate, or yeast sources) 4 mg,vitamin B2 (riboflavin, or yeast sources) 5 mg,vitamin B3 (niacinamide or yeast sources) 20 mg,vitamin B6 (pyridoxine hydrochloride or yeast sources) 5 mg,folic acid (folate, yeast source, or liver source) 800 mcg,vitamin B12 (cyanocobalamin or yeast source) 10 mcg,biotin (d-biotin or liver sources) 200 mcg,pantothenic acid (D-calcium pantothenate, yeast, rice bran, or liversources) 10 mg,calcium (citrate, ascorbate, plant based sources or lactate) 250 mg,magnesium (citrate, lactate, or natural forms) 125 mg,selenium (seleno-L-methionine, or natural forms) 100 mcg,zinc (glycinate or natural forms) 15 mg,chromium (picolinate or natural forms) 50 mcg, andomega-3 fatty acids (EPA:DHA, 3:2) 2000 mg.

Example 3

A “booster” formulation, for ages 14 years and older, is provided in apackage form (one capsule plus one fatty acid gel-cap), liquid form (onepacket dissolved in liquid), beverage bottle (2-8 ounces) or bar form(one bar) and is intended to be taken once as a separate“neuroprotective” package one to two hours prior to engaging inpotentially violent contact sports, undertaking anoccupationally-related hazardous activity where the risk of concussiveinjury is increased, or, for active military personnel, entering acombat or hazardous duty zone.

The preparation is comprised of:

vitamin E (d-alpha tocopheryl succinate, vegetable products, naturaltocopheryl, or wheat germ products) 50 IU,vitamin C (calcium ascorbate, citrus, rose, or berry products) 200 mg,coenzyme Q10 (ubiquinone) 25 mg,N-acetyl cysteine 100 mg,acetyl L-carnitine (fumarate) 100 mg, andomega-3 fatty acids (EPA:DHA, 3:2) 1000 mg.

Example 4

A “protection” formulation, ages 8 years older, is provided in a packageform (one capsule plus one fatty acid gel-cap taken twice per day),liquid form (one packet dissolved in liquid twice per day), or bar form(one bar consumed twice per day) and is intended to be taken for thetotal number of days as directed by health care professional for thosewho have sustained a concussive brain injury.

The preparation is comprised of:

vitamin E (d-alpha tocopheryl succinate, vegetable products, naturaltocopheryl, or wheat germ products) 50 IU,vitamin C (calcium ascorbate, citrus, rose, or berry products) 100 mg,coenzyme Q10 (ubiquinone) 45 mg,N-acetyl cysteine 50 mg,acetyl L-carnitine (fumarate) 50 mg, andomega-3 fatty acids (EPA:DHA, 3:2) 500 mg.

Doses in the above examples are recommended but can be flexible. Dosesfor example formulations 1, 2, & 4 should be taken at least once perday. Example formulations are target doses, but doses may vary withinabove ranges.

The micronutrient supplements should be taken orally and divided intotwo doses, half in the morning and the other half in the eveningpreferably with meals. This is because the biological half-lives ofmicronutrients (water-soluble and fat-soluble) are highly variable whichcan create marked fluctuations in tissue levels, differences in geneprofile expression and cellular stress. This is also important tomaintain relatively consistent levels of micronutrients in the brain.

No iron, copper or manganese would be included because these traceminerals are known to interact with vitamin C to produce free radicals.These minerals are also absorbed more readily from the intestinal tractin the presence of antioxidants, and this could result in potentiallyharmful increased body stores of unbound minerals. Increased iron storeshave been linked to a higher risk of several chronic diseases (121).

Evidence of Effectiveness of Micronutrient Formulations 1) OxidativeDamage Impact:

Prospective, randomized, double-blind, placebo-controlled trial in 34U.S. Marine Corps 1^(st) Tank volunteers subjected to cold, highaltitude and exertion stress at the Mountain Warfare Training Center, 29Palms, Calif. (122).

The study group consumed the PMC formulation and the control groupreceived a placebo supplement for duration of the 12 week trainingcourse. Serial urine samples were taken from all participants andanalyzed before and after supplementation for sensitive markers ofoxidative damage (8-hydroxyguanosine). Safety was assessed by fieldreports of adverse reactions.

In the placebo group, 42% of subjects had low levels of the biologicalmarker before supplementation, and the remaining 58% exhibited highlevels of oxidative damage reflecting the extreme conditions. Afterconsuming the placebo, only 25% of the subjects still had low levels ofoxidative damage and 75% had high levels, demonstrating 17%deterioration in oxidative status.

Conversely, in the antioxidant treated group, 30% of the subjects hadlow levels of 8-hydroxyguanosine before supplementation and 70%exhibited high levels of the marker. After receiving the PMCformulation, 71% of the subjects showed low oxidative damage levels andonly the remaining 29% had high levels, demonstrating 41% improvement inoxidative status (table). This documented that the formulation not onlyprevented more oxidative stress from occurring during extreme exercisebut, in fact, reduced the oxidative damage that was already present.This significant recovery in the treatment group showed a highlydesirable impact during military training and translates directly to theexertion and stress experienced in elite or recreational sports, andhazardous occupations.

In addition, the changes in plasma levels of antioxidant micronutrientswere also documented. The placebo group showed no difference in thelevels of alpha-tocopherol (vitamin E) in the blood, before (4.8 ug·ml)or after (4.6 ug/ml) supplementation. However, the average value almostdoubled (4.1 ug/ml to 7.6 ug/ml) in the antioxidant treated group,documenting that the PMC formulation is absorbed well even in the faceof extreme conditions and intense exercise. There were no reported orobserved adverse effects from consuming the formulation.

TABLE 1 Military Antioxidant Placebo Formulation Post- Post-Pre-treatment treatment Pre-treatment treatment N = 12 N = 12 N = 17 N =14 (% of total subjects) (% of total subjects) Low value 42 25 30 71High value 58 75 70 29

The 8-hydroxuguanosine level (μg/mg creatine) up to 2.0 was consideredlow value (low oxidative damage) and 2.1 to 3.0 as high value (highoxidative damage).). The number of subjects with high oxidative damageincreased after placebo consumption. However, the number of subjectswith high oxidative damage decreased after antioxidant treatment. Nrefers to the number of subjects in each group.

2) Beneficial Impact after Concussive Brain Injury:

Prospective, randomized, double-blind clinical trial in 42 U.S. MarineCorps personnel suffering TBI from moderate concussive brain injury(blasts) after returning from war in Iraq (123). All patients hadreceived their injury 3-20 weeks prior to study entry. The control groupreceived standard rehabilitation (steroids, physical therapy, vestibularrehabilitation and supportive care) for 12 weeks. The study groupreceived standard care plus the formulation (two capsules by mouth twiceper day) for the same time period.

All patients were evaluated by the same outcome measures that includedthe Sensory Organization Test (SOT) by Computerized DynamicPosturography (CDP), Dynamic Gait Index (DGI), the Activities BalanceConfidence (ABC) scale, Dizziness Handicap Index (DHI), VestibularDisorders Activities of Daily Living (VADL) score, and the BalanceScoring System (BESS) test. The therapist who performed and graded thetesting was blinded as to whether or not the patient was in the controlgroup or receiving antioxidant therapy. The pre-trial test scores didnot differ significantly between the two groups on any of the tests.

The study group receiving the micronutrient formulation demonstratedmore rapid and complete recovery than did the control group even thoughthe formulation was not consumed until well after the concussions weresuffered. Postural stability, dynamic gait index, and dizziness handicapscores were already significantly different after only 4 weeks, and thisimprovement grew in significance by the end of 12 weeks. The SOT scoreby CDP was 78 for the antioxidant group as compared to 63 for thecontrol group (P<0.05).

The improvement noted in the micronutrient group on the other tests alsotrended to a greater degree than that of the control group.Questionnaires were also administered regarding energy levels, exercise,and overall cognitive issues by an investigator who did not know towhich group the patient belonged. The study group demonstrated asignificant increase in energy level, exercise tolerance and cognitiveability at every weekly time point. There were no adverse effects fromthe PMC formulation.

3) Prevention of Neurological Injury:

This was a laboratory study of Parkinson's disease in a validated rodentmodel (124). The agent, MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), is a contaminant incertain recreational drugs and can induce Parkinson's disease in humans.This substance can also be administered intraperitoneally in rats andinduces severe Parkinsonian symptoms allowing this model to be used tounderstand the mechanisms of the disease.

The PMC formulation was fed to the study group prior to administrationof MPTP and provided a significant degree of neuroprotection compared tothe control group. The supplemented rodents demonstrated an 86%reduction in the development of Parkinson's disease compared to the ratsthat received only standard diets.

4) Prevention of Concussion-Related Inflammation

This was a laboratory study of concussive blast overpressure in avalidated rodent model (125). The study formulation was administeredorally to rats for 7 days before exposure to a whole-body concussiveblast dose of 150 kilopascals. This is generally a lethal overpressuredose in rodents, and represents a potentially concussion-producing blastin humans, both of which would cause a significant rise in markers ofsevere inflammation in the bloodstream.

The supplementation demonstrated a dramatic protective effect in that itcompletely prevented the rise of inducible nitric oxide synthase, acritical enzymatic marker of the inflammatory cascade in the blood ofthe animals. In addition, the supplementation did not adversely affectcertain enzymatic markers of oxidative damage such as hemeoxygenase-1and superoxide dismutase.

The formulations in this application support tissue repair and recoveryduring exertion and extreme conditions as related to oxidative damageand inflammation. The validating studies, now published in thepeer-reviewed clinical medical literature (126), show beneficial impacton concussive events, both preventatively before an event occurs, andsupportively after an event is suffered.

In addition to the risk of TBI, high intensity activity in sports,occupational or military environments is also associated with enormousproduction of excess free radicals and results in measurable oxidativedamage to the body's cells and tissues. This type of effort also causessignificant inflammatory responses in the body and is known to suppressnormal immune function. Specific formulations based on biochemicalmechanisms in concussive brain injury and exploiting the knownantioxidant science have been developed to addresses the risks from thisspectrum of violent activity.

Numerous modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the attendant claims attachedhereto, this invention may be practiced otherwise than as specificallydisclosed herein.

1. A selection method for separating target cells from non-target cellsin a sample comprising target cells, secondary targets and non-targetcells, the method comprising: a) contacting the sample with a firstantibody composition comprising (a) at least one antibody that binds tothe target cells linked, either directly or indirectly, to (b) at leastone antibody that binds to the secondary targets, under conditions toallow immunorosettes of the target cells and the secondary targets toform; b) contacting the sample with a second antibody compositioncomprising (a) at least one antibody that binds to the secondary targetseither singly or within the immunorosettes, linked, either directly orindirectly, to (b) at least one antibody that binds to particles, underconditions to allow binding of the particles to the immunorosettesand/or the secondary targets, and c) separating theimmunorosette—particle complexes and/or the secondary target—particlecomplexes from the sample to separate the target cells from thenon-target cells.
 2. The method of claim 1 wherein the second antibodycomposition is bound to the particles prior to contacting with thesample.
 3. The method of claim 1 wherein the sample is contacted with aparticle bound to antibodies that bind to the secondary target.
 4. Themethod of claim 1 wherein the second antibody composition is bound tothe particles and added to the sample prior to contacting the samplewith the first antibody composition.
 5. The method of claim 1, whereinthe secondary targets are erythrocytes, granulocytes, or beads,optionally polystyrene beads. 6-7. (canceled)
 8. The method of claim 5,wherein the polystyrene beads are coated with a polymer.
 9. The methodof claim 1, wherein the secondary targets are added to the sample priorto separation.
 10. The method of claim 1, wherein the particles aremagnetic particles.
 11. The method of claim 1, wherein the particles arenon-magnetic particles.
 12. The method of claim 1, wherein the selectionmethod is a positive selection method to select for a desired cell andthe target cell is the desired cell or a negative selection method toremove a non-desired cell from the sample and the target cell is thenon-desired cell.
 13. (canceled)
 14. The method of claim 1, wherein thesecondary targets have a density similar to a cell and the particleshave a density different from a cell.
 15. The method of claim 1, whereinthe immunorosette-magnetic particle complexes and/or the secondarytarget-magnetic particle complexes are separated from the sample in stepc) by placing the sample in a magnetic field gradient of sufficientstrength to separate the immunorosette-magnetic particle complexesand/or the secondary target-magnetic particle complexes from the sample.16. The method of claim 1, wherein the immunorosette-magnetic particlecomplexes are separated from the sample in step c) by density separationor by sedimentation.
 17. (canceled)
 18. The method of claim 12, whereinthe method further comprises: d) separating the target cells from theimmunorosette-magnetic particle complexes.
 19. The method of claim 14,wherein the target cells are separated from the immunorosette-magneticparticle complexes by physical, chemical, enzymatic or thermaldissociation.
 20. The method of claim 5, wherein the method furthercomprises: d) lysis of the erythrocytes in the immunorosettes and e)separation of the target cells from the lysed erythrocytes and themagnetic particles.
 21. The method of claim 1, wherein the sample isblood, whole blood, bone marrow, fetal liver, cord blood, a buffy coatsuspension, a leukapheresis sample, a pleural and periotoneal effusionor a sample of thymocytes and splenocytes.
 22. The method of claim 1,wherein the target cells are stem cells, progenitor cells, monocytes,lymphocytes (such as T cells, B cells or NK cells) or granulocytes (suchas neutrophils, basophils or eosinophils). 23-24. (canceled)
 25. Themethod according to claim 1 wherein the first antibody composition is abi-specific tetrameric complex and the second antibody composition is amono-specific tetrameric complex.
 26. (canceled)
 27. The method of claim19 wherein the mono-specific tetrameric complex comprising antibodiesthat bind to erythrocytes.